Nucleic acid sequences showing enhanced expression in benign neuroblastoma compared with acritical human neuroblastoma

ABSTRACT

There are disclosed a nucleic acid which is derived from the gene expressed in human neuroblastoma, and which comprises any sequence selected from the group consisting of the nucleic acid sequences set forth SEQ ID NO:1 to NO:104 in the Sequence Listing, or its complementary nucleic acid; a fragment of the nucleic acid; their use as probes or primers; and the diagnosis of neuroblastoma prognosis using any of the foregoings.

CROSS-REFERENCED APPLICATIONS

This application is a National phase of International ApplicationPCT/JP01/01631, filed Mar. 2, 2001, which designated the U.S. and thatInternational Application was not published under PCT Article 21(2) inEnglish.

TECHNICAL FIELD

This invention relates to nucleic acids derived from genes expressed inhuman neuroblastomas. More specifically, the invention relates tonucleic acids and their fragments derived from the genes whoseexpression is enhanced in human neuroblastoma with favorable prognosisbased on comparison between human neuroblastoma with favorable prognosisand human neuroblastoma with unfavorable prognosis as well as to theirutility in the diagnosis of prognosis for human neuroblastomas.

BACKGROUND ART

Individual tumors exhibit distinct characteristic natures, and theirbiological properties are not necessarily identical even though thebasic principle of oncogenesis is the same. Rapid advances in theunderstanding of cancer from a molecular biological and moleculargenetic perspective in recent years have opened the way to anexplanation of oncogenesis and tumor cell biology on the genetic level.

(Neuroblastomas)

Neuroblastoma is a pediatric cancer occurring in sympatheticgangliocytes and adrenal medullary cells which originate from cells ofthe peripheral sympathetic nervous system. Of these sympathetic nervoussystem cells, neural crest cells in the initial stage of developmentmigrate to the abdomen, differentiating and maturing at sites wheresympathetic ganglia are formed. Some of these cells migrate further tothe adrenal bodies, penetrating through the adrenal cortex which isalready in the process of formation, and reaching the medulla andforming medullary substance there. The neural crest cells also serve asa source of other peripheral nerve cells, differentiating into dorsalroot ganglia (sensory nerves), skin pigment cells, thyroid C cells, somepulmonary cells, intestinal gangliocytes, and the like.

(Prognosis for Neuroblastoma)

Neuroblastoma is characterized by a varied clinical profile (Nakagawara,Shinkeigashu no Hassei to Sono Bunshi Kiko [Neuroblastoma Developmentand Molecular Mechanism], Shoni Naika 30, 143, 1998). For example,neuroblastomas occurring at less than one year of age have veryfavorable prognosis, with the majority undergoing differentiation andcell death, and spontaneous regression. Currently, most neuroblastomasdiscovered by a positive result in the commonly performed mass screeningof 6-month-old infant urine are of the type which tend to undergo thisspontaneous regression. On the other hand, neuroblastomas occurring atage 1 or higher are highly malignant and lead to death of the infant inthe majority of cases. It is also hypothesized that a somatic mutationoccurs in highly malignant neuroblastomas in infants older than one yearof age, which are of monoclonal nature, whereas in naturally regressingneuroblastomas, the genetic mutation remains at only a germlinemutation. See Knudson A G, et al.: Regression of neuroblastoma IV-S: Agenetic hypothesis, N. Engl. J. Med. 302, 1254 (1980)).

(Tumor Markers which Allow the Diagnosis of Prognosis for Neuroblastoma)

With recent advances in molecular biology research, it has become clearthat expression of the high affinity nerve growth factor (NGF) receptorTrkA is closely connected with control of differentiation and celldeath. See Nakagawara A., The NGF story and neuroblastoma, Med. Pediatr.Oncol., 31, 113 (1998). Trk is a membrane-spanning receptor, existing asthe three main types, Trk-A, -B and -C. These Trk family receptors playan important role in specific nerve cell differentiation and survival inthe central nervous and peripheral nervous systems. See Nakagawara, etal., Shinkeigasaiboushu ni Okeru Neurotrophin Juyoutai no Hatsugen toYogo [Expression of Neurotrophin Receptors and Prognosis inNeuroblastoma], Shoni Geka (Pediatric Surgery), 29:425–432, 1997. Thesurvival and differentiation of tumor cells is controlled by signalsfrom Trk tyrosine kinase and Ret tyrosine kinase. In particular, therole of TrkA receptor is most significant, with TrkA expression beingnotably high in neuroblastomas with favorable prognosis, and its signalsexerting a powerful control over survival and differentiation of tumorcells, and cell death (apoptosis). In neuroblastomas with unfavorableprognosis, on the other hand, TrkA expression is significantlysuppressed, while tumor development is aided by a mechanism in whichsurvival is promoted by signals from TrkB and Ret.

It has become clear that amplification of the neural oncogene N-myc hasbecome clearly associated with the prognosis of neuroblastoma. SeeNakagawara, Nou-shinkeishuyo no Tadankai Hatsugan [MultistageOncogenesis of Cerebral and Neural Tumors], Molecular Medicine, 364,366(1999). This gene, first cloned in neuroblastoma, is ordinarily onlypresent in a single copy per haploid set in normal cells andneuroblastomas with favorable prognosis, whereas it has been found to beamplified several dozen times in neuroblastomas with unfavorableprognosis. Thus, amplification of N-myc is closely linked to tumorprogression.

Up till the present time, however, no oncogene other than N-myc is knownto be expressed in neuroblastomas, and absolutely no genetic informationother than that of N-myc has been known in relation to favorable orunfavorable prognosis.

DISCLOSURE OF THE INVENTION

This invention has been accomplished in light of the circumstancesdescribed above, and its object is to identify the information of geneswhich are expressed in neuroblastomas, to further identify theinformation of the genes which is related to favorable or unfavorableprognosis, and to allow the diagnosis for favorable or unfavorableprognosis of neuroblastoma based on that genetic information.

In the course of conducting diligent research in line with theaforementioned object, the present inventors have examined the prognosesof neuroblastomas and have succeeded in constructing cDNA libraries fromclinical tissues with favorable prognosis and unfavorable prognosis.Approximately 2400 clones were respectively obtained from these twotypes of cDNA libraries and were classified according to the prognosisof neuroblastoma (whether favorable or unfavorable).

The present inventors further determined the partial or whole sequencesof these cloned genes, and upon performing a homology search, selectedsuitable genes.

Moreover, upon comparing the classified gene groups as described aboveagainst the selected genes, the present inventors found that theexpression of a considerable number of the genes is enhanced only inclinical tissues of neuroblastoma with favorable prognosis.

Based on such knowledge, the present inventors have succeeded inproviding genetic information (nucleic acid sequence information etc.)for the detection and cloning of the genes only expressed in humanneuroblastomas with favorable prognosis. Furthermore, based on theaforementioned nucleic acid sequence information it has been madepossible to carry out the method for detection of prognosis and todesign tumor markers which can be used therefor, and this invention hasthereupon been completed.

Specifically, this invention provides the nucleic acids and nucleic acidfragments described under 1. to 8. below. The invention further providesuses for those nucleic acids and nucleic acid fragments as describedunder 9 to 11. below.

1. A nucleic acid derived from a gene expressed in human neuroblastoma,the nucleic acid comprising a sequence selected from the groupconsisting of the nucleic acid sequences set forth in SEQ ID NO:1 toNO:104 in the Sequence Listing, or its complementary nucleic acid.

2. The nucleic acid according to 1. above, characterized in that thenucleic acid is DNA.

3. A nucleic acid derived from a gene whose expression is enhanced inhuman neuroblastoma with favorable prognosis based on comparison betweenhuman neuroblastoma with favorable prognosis and human neuroblastomawith unfavorable prognosis, the nucleic acid comprising a sequenceselected from the group consisting of the nucleic acid sequences setforth in SEQ ID NO:1 to NO:104 in the Sequence Listing, or itscomplementary nucleic acid.

4. The nucleic acid according to 3. above, characterized in that thenucleic acid is DNA.

5. A fragment of the nucleic acid according to any one of 1. to 4.above.

6. An isolated nucleic acid which can hybridize to the nucleic acidaccording to any one of 1. to 4. above under stringent conditions.

7. The isolated nucleic acid according to 6. above, characterized inthat the nucleic acid is DNA.

8. A PCR primer comprising the nucleic acid according to 7. above.

9. A method of diagnosing the prognosis of human neuroblastoma, themethod comprising detecting the nucleic acid according to 3. above fromclinical tissue of human neuroblastoma.

10. A diagnosis kit for the prognosis of human neuroblastoma, containinga pair of PCR primers according to 8. above.

Accordingly, preferred as the nucleic acid described above is nucleicacid derived from a gene whose expression is enhanced only in humanneuroblastoma with favorable prognosis, upon comparison between humanneuroblastoma with favorable prognosis and human neuroblastoma withunfavorable prognosis, as information relating to the sequence of saidnucleic acid will allow the diagnosis for prognosis of humanneuroblastoma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration corresponding to an electrophoregram showingan example of a gene whose expression was found enhanced in humanneuroblastomas with favorable prognosis (the result from nucleic acidsequence nbla-00106), as a result of examining the level of geneexpression in human neuroblastomas with favorable prognosis and withunfavorable prognosis by semi-quantitative PCR. In the figure, Lanes1–16 are clinical tissue specimens of human neuroblastomas withfavorable prognosis. On the other hand, Lanes 17–32 are clinical tissuespecimens of human neuroblastomas with unfavorable prognosis.

FIG. 2 is an illustration corresponding to an electrophoregram showinganother example of a gene whose expression was found enhanced in humanneuroblastomas with favorable prognosis (the result from nucleic acidsequence nbla-00219), as a result of examining the level of geneexpression in human neuroblastomas with favorable prognosis and withunfavorable prognosis by semi-quantitative PCR. In the figure, Lanes1–16 are clinical tissue specimens of human neuroblastomas withfavorable prognosis. On the other hand, Lanes 17–32 are clinical tissuespecimens of human neuroblastomas with unfavorable prognosis.

FIG. 3 is an illustration corresponding to an electrophoregram showingstill another example of a gene whose expression was found enhanced inhuman neuroblastoma with favorable prognosis (the result from nucleicacid sequence nbla-03145), as a result of examining the level of geneexpression in human neuroblastomas with favorable prognosis and withunfavorable prognosis by semi-quantitative PCR. In the figure, Lanes1–16 are clinical tissue specimens of human neuroblastomas withfavorable prognosis. On the other hand, Lanes 17–32 are clinical tissuespecimens of human neuroblastomas with unfavorable prognosis.

FIG. 4 is an illustration corresponding to an electrophoregram showingan example of a gene whose expression was noted (the result from nucleicacid sequence nbla-00100), as a result of examining the level of cellcycle phase-specific gene expression by semi-quantitative PCR. In thefigure, Lane 1 represents untreated HeLa cells (60–70% confluent). Lane2 represents HeLa cells treated with 400 μM of mimosine for 18 hours,with 65% arrested in the G1 phase. Lane 3 represents HeLa cells treatedwith 2 mM thymidine for 20 hours, with 100% arrested in the S phase.Lane 4 represents HeLa cells treated with 0.6 μg/ml of nocodazole for 18hours, with 85% arrested in the G2/M phase.

BEST MODE FOR CARRYING OUT THE INVENTION

The nucleic acids derived from the genes expressed in humanneuroblastomas according to this invention (hereinafter referred to as“genes of the invention”) and their related nucleic acid fragments(hereinafter referred to respectively as “nucleic acids of theinvention” and “nucleic acid fragments of the invention”, or wheredistinction between the nucleic acids and their fragments is notparticularly necessary in description, they will be collectivelyreferred to as “nucleic acids of the invention”) will now be explainedin greater detail, with reference to preferred embodiments of theinvention.

The nucleic acids of the invention are derived from the genes of theinvention as mentioned above, and they either constitute the genes orare obtained from the genes by in vivo or in vitro procedures. The term“nucleic acids” as used throughout the present specification refers to,for example, DNA or RNA, or polynucleotides derived therefrom which areactive as DNA or RNA, and preferably they are DNA or RNA. Particularlypreferred nucleic acids either have sequences identical to the humancDNA sequences disclosed in the present specification or have sequencescomplementary thereto.

The term “hybridize under stringent conditions” as used in the presentspecification means that two nucleic acids (or fragments) hybridize toeach other under the hybridization conditions described by Sambrook, J.et al. in “Expression of cloned genes in E. coli”, Molecular Cloning: ALaboratory Manual (1989), Cold Spring Harbor Laboratory Press, New York,USA, 9.47–9.62 and 11.45–11.61.

More specifically, the “stringent conditions” refers to hybridization atapproximately 45° C., 6.0×SSC, followed by washing at 50° C., 2.0×SSC.The stringency may be selected by choosing a salt concentration in thewashing step from approximately 2.0×SSC, 50° C. as low stringency toapproximately 0.2×SSC, 50° C. as high stringency. Also, the temperaturein the washing step may be increased from room temperature, orapproximately 22° C. as low stringency conditions, to approximately 65°C. as high stringency conditions.

The term “isolated nucleic acid” as used throughout the presentspecification refers to a nucleic acid or a polynucleotide containingsubstantially no cellular substances or culture medium, if prepared byrecombinant DNA techniques, or containing substantially no precursorchemical substances or other chemical substances, if prepared bychemical synthesis.

The term “favorable prognosis” as used throughout the presentspecification refers to a condition of human neuroblastoma in which thetumor is localized or has become a regressing or benign sympatheticganglion neoplasm, and is judged by a physician to have low malignancybased on N-myc or other tumor markers (TrkA, chromosomal aberration,etc.). According to a preferred embodiment of the invention, a favorableprognosis is a case of stage 1 or 2, with an onset age of less than oneyear and survival without recurrence for 5 or more years after surgery,and with no amplification of N-myc in the clinical tissue; however,there is no limitation to such specific cases. The term “unfavorableprognosis” as used throughout the present specification refers to acondition of human neuroblastoma in which progression of the tumor hasbeen observed, and it is judged by a physician to have high malignancybased on N-myc or other tumor markers. According to a preferredembodiment of the invention, an unfavorable prognosis is a case of stage4, with an onset age of greater than one year, death within 3 yearsafter surgery and amplification of N-myc in the clinical tissue;however, there is no limitation to such specific cases.

Neuroblastoma is a tumor consisting of actual nerve cells, of which onlytwo types of tumor are known in humans, and analysis of the genesexpressed therein is expected to provide very useful knowledge forunderstanding the biology of nerve cells. Specifically, it is extremelydifficult, and practically impossible, to obtain site-specifichomogeneous tissue from the brain or peripheral nerves. On the otherhand, a neuroblastoma consists of an almost homogeneous nerve cellpopulation (though tumorized) derived from peripheral sympathetic nervecells, and thus offers a high possibility of obtaining homogeneousexpression of neuro-related genes. Furthermore, since neuroblastoma is atype of cancer, it will characteristically have many important genesexpressed in the immature stage of neurogenesis.

Clinically and biologically, neuroblastoma can be neatly classified intofavorable prognosis and unfavorable prognosis types. Cancer cells fromneuroblastoma with favorable prognosis are characterized by having avery slow rate of proliferation, with spontaneous regression beginningat some point. Findings to date have confirmed that nerve celldifferentiation and apoptosis (nerve cell death) occur in thespontaneous regression, and that the differentiation which occurs in thematuration stages of normal nerve cells and programmed cell death arephenomena very closely resembling each other. Consequently, it is highlyprobable that the analysis of genes expressed in such tumors will leadto obtaining important genetic information relating to nerve celldifferentiation and apoptosis.

Neuroblastomas with unfavorable prognosis are tumors consisting ofcancer cells which continue to exhibit definitely malignantproliferation. The probability is very high, therefore, that they have alarge number of important genes connected with nerve cell proliferationor genes expressed in undifferentiated nerve cells. In other words, itis highly probable that these will allow the obtainment of geneticinformation completely different from the profile of genes expressed inneuroblastomas with favorable prognosis.

It is commonly reported that nerve cells contain more expressed genetypes than cells derived from other organs. Neuroblastoma cell lines arederived from clinical tissues with unfavorable prognosis, and it isbelieved that the gene expression profile in the case of tumordevelopment and progression is substantially altered from that of normalnerve cells.

Neuroblastoma is characteristically a pediatric tumor, and because ofthe very low possibility of effects by acquired factors, it is expectedthat analysis of the mechanism of cancerization will also yieldembryological information with high probability. More surprisingly, thenucleic acids of the invention include nucleic acids of genes whoseexpression is enhanced only in specific cell cycle phases, and thisfurther suggests the very strong possibility of obtaining geneticinformation highly useful for the analysis of cancerization mechanismsand related to development and differentiation.

The nucleic acids of the invention, having the various characteristicsmentioned above and derived from the genes which can yield usefulgenetic information, are obtained from human neuroblastoma clinicaltissues and have any of the nucleic acid sequences set forth in SEQ IDNO:1 to NO:104 in the Sequence Listing, or a portion thereof.

As a result of comparing levels of expression of the genes according tothis invention in clinical tissues from human neuroblastomas withfavorable prognosis and with unfavorable prognosis, a highly significantdifference was found in the genes corresponding to the nucleic acidsequences set forth I SEQ ID NO:1 to NO:104 in the Sequence Listing.That is, expression of these genes was enhanced in human neuroblastomaswith favorable prognosis. Thus, in addition to providing the usefulgenetic information described above, the nucleic acid sequences setforth in SEQ ID NO:1 to NO:104 can also be utilized as data for tumormarkers to diagnose favorable or unfavorable prognosis of neuroblastoma,by detecting the nucleic acid having any of these nucleic acidsequences.

Specifically, this invention will make it possible to obtain variousgene information on or relating to human neuroblastoma through thefollowing means.

(1) Probes for Hybridization

According to one embodiment of this invention, the nucleic acids of theinvention or their fragments may be used as probes for hybridization inorder to detect genes expressed in human neuroblastoma. The nucleicacids of the invention or their fragments may also be used as probes forhybridization in order to determine gene expression in various tumorsand normal tissues, to identify the distribution of the gene expression.

When the nucleic acids of this invention or their fragments are used asprobes for hybridization, there are no particular limitations on theactual method of hybridization. As preferred methods there may bementioned, for example, Northern hybridization, Southern hybridization,colony hybridization, dot hybridization, fluorescence in situhybridization (FISH), in situ hybridization (ISH), DNA chip methods, andmicroarray methods.

As one application example of the hybridization, the nucleic acid ofthis invention or its fragment may be used as a probe for Northernhybridization to measure the length of mRNA or to quantitatively detectgene expression in a sample to be examined.

As another application example, the nucleic acid of the invention or itsfragment may be used as a probe for Southern hybridization to detect thepresence or absence of the DNA sequence in genomic DNA of a sample to beexamined.

As still another application example, the nucleic acid of the inventionor its fragment may be used as a probe for fluorescence in situhybridization (FISH) to identify the location of the gene on achromosome.

As yet another application example, the nucleic acid of the invention orits fragment may be used as a probe for in situ hybridization toidentify the tissue distribution of gene expression.

When the nucleic acid of the invention or its fragment is used as aprobe for hybridization, a nucleic acid residue length of at least 40 isnecessary; and among the nucleic acids and their fragments of theinvention, the one with 40 or more contiguous residues or its fragmentis preferably used. More preferably, the one with 60 or more residues isused.

Nucleic acid probe techniques for the types of hybridization mentionedabove are well known to one skilled in the art, and for example,conditions suitable for hybridization between a nucleic acid probe ofvarious lengths according to the invention and target polynucleotide maybe readily determined. For example, Sambrook et al. described in“Molecular Cloning: A Laboratory Manual, loc. cit. may be followed forsuch manipulations which are well known to one skilled in the art.

A probe according to this invention is preferably labeled in an easilydetectable fashion. The detectable label may be an element or compound,of any type which can be detected either visually or using devices. Ascommonly used detectable labels there may be mentioned radioactiveisotopes, avidin or biotin, and fluorescent substances (FITC, rhodamine,and the like). The radioactive isotopes include ³²P, ¹⁴C, ¹²⁵I, ³H and³⁵S. Biotin-labeled probes are detected after hybridization usinglabeling means such as avidin/streptavidin, fluorescent labels, enzymes,gold colloidal complexes or the like. A probe according to the inventionmay also be labeled by binding with a protein. Radioactive orfluorescent histone single-stranded DNA binding protein may be used forthis purpose.

(2) Primers for use in PCR

In addition to hybridization for the detection of target genes (e.g.,the genes according to this invention), any nucleic acid sequenceincluded in the nucleic acid of the invention or its fragment may beused as a primer in a polymerase chain reaction (PCR). For example, mRNAmay be extracted from a sample to be examined, and the gene expressionmay be semi-quantitatively measured by RT-PCR. This may be carried outby a method well known to one skilled in the art. See, for example,Sambrook et al. described in “Molecular Cloning: A Laboratory Manual,”loc. cit. and Idenshibyo Nyumon [Introduction to Genetic Diseases](Takahisa, S.: Nankodo Publishing).

When the nucleic acid of this invention or its fragment is used as a PCRprimer, a nucleic acid residue length of 10 to 60 is necessary; andamong the nucleic acids of the invention and their fragments, the onewith 10 to 60 contiguous residues or its fragment is preferably used.More preferably, the one with 15 to 30 residues is used. In most cases,a primer sequence with a GC content of 40–60% is preferred. Also, thereis preferably no difference in the Tm values of the two primers used foramplification. Preferably there is no annealing at the 3′ ends of theprimers and no secondary structure is formed in the primers.

(3) Gene Screening

A nucleic acid of the invention or its fragment may also be used todetect the expression distribution of a target gene which is expressedin various tissues or cells. This may be accomplished, for example, byusing the nucleic acid of the invention or its fragment as a probe forhybridization or as a primer for PCR, as described above.

Expression distribution of a target gene can also be detected using aDNA chip, microarray or the like. That is, the nucleic acid of theinvention or its fragment may be directly attached to the chip or array.For this purpose, methods for spotting such a nucleic acid (DNA) ontoplates using a high precision dispenser are known (see, for example,U.S. Pat. No. 5,807,522). mRNA extracted from cells of a tissue specimenmay be labeled there with a fluorescent substance or the like,hybridized, and an analysis may be made of the type of tissue cells withhigh expression of the gene. The DNA attached to the chip or array maybe the reaction product of PCR using the nucleic acid of the inventionor its fragment as the primer. Alternatively, nucleic acid fragments ofthe invention may be directly synthesized on a substrate to prepare aDNA chip or array (see, for example, U.S. Pat. No. 5,424,186).

(4) Gene Cloning

The nucleic acid of this invention or its fragment may be used forcloning a gene which is expressed in human neuroblastoma. For example,by using the nucleic acid of the invention or its fragment as a probefor northern hybridization or southern hybridization, or as a primer forPCR, cloning of a gene containing the nucleic acid of the invention orits fragment is possible. As the genes subjected to such cloning theremay be mentioned genes with differing levels of expression particularlybetween neuroblastoma with favorable prognosis and neuroblastoma withunfavorable prognosis, genes whose forms of expression differ in othertissues or cancer cells, genes whose expression is cell cyclephase-dependent, genes induced upon neurodifferentiation and genes whoseexpression is regulated by oncogenes or tumor suppressor genes. Thecloning may be carried out according to ordinary gene recombinationtechniques, by incorporating the nucleic acid of the invention or itsfragment into an appropriate plasmid or bacteriophage to construct anexpression vector, introducing this into host cells for transformation(or transduction), and culturing the transformants. The individualmanipulations for this procedure are described in detail by Sambrook etal. in “Molecular Cloning: A Laboratory Manual,” loc. cit., as well asin other well-known literature.

(5) Methods of Diagnosing Tumor Prognosis and Tumor Markers to be UsedTherefor

As mentioned above, the genes related to the nucleic acids of thisinvention have their expression enhanced in human neuroblastomas withfavorable prognosis. The nucleic acid (DNA) of the invention or itsfragment may therefore be used as a probe for hybridization, or as aprimer for PCR to allow the identification of prognosis. Specifically,this may be accomplished by examining whether the expression of the geneis enhanced in a clinical tissue containing sample taken from a subject.The methods of detecting the gene include Northern blottinghybridization, in situ hybridization and RT-PCR, as mentioned aboveamong others.

When hybridization is employed, prognosis may be diagnosed as favorableif the amount of nucleic acid hybridizing to the probe is increased inthe sample. When RT-PCR is employed, mRNA is extracted from the sampleand reverse transcribed into DNA, amplification is performed using theaforementioned primer, and the gene expression is semi-quantitativelymeasured. The prognosis may be diagnosed as favorable if the geneexpression is then found to be enhanced. For the purpose of suchspecific diagnosis it is preferred to utilize a diagnosis kit containinga pair of such primers as essential components. In addition to theprimer components, the diagnosis kit also include known components suchas PCR buffer, detergent solution and enzymes.

(6) Antisense Oligonucleotides

According to another embodiment of this invention there are providedantisense oligonucleotides to the nucleic acids of the invention. Theantisense oligonucleotides are capable of hybridizing to the nucleicacids of the invention, and include antisense DNAs and antisense RNAS.Antisense DNA inhibits transcription of mRNA from DNA, while antisenseRNA inhibits translation of mRNA. Native types of such antisenseoligonucleotides may be synthesized using an automated synthesizer or byPCR using the nucleic acid of the invention as templates. The antisenseoligonucleotides also encompass antisense oligonucleotide derivativeshaving improved binding affinity for the target DNA or mRNA, tissueselectivity, cell permeability, nuclease resistance and intracellularstability. Such derivatives may be synthesized using antisensetechnology known in the art.

Antisense oligonucleotides having sequences complementary to thesequences near the translation initiation codon of the mRNA, those ofthe ribosome-binding site, and those of the capping site or the splicingsite are capable of inhibiting synthesis of the RNA and therefore willexhibit a particularly notable inhibitory effect on gene expression.This invention therefore encompasses such antisense oligonucleotides.

(7) Gene Therapy

According to a further embodiment of this invention, there are providednucleic acid sequences encoding the therapeutic genes to be used in genetherapy. Thus, the nucleic acid of the invention can be transferred intoa vector for use in gene transportation, whereby the transgene (i.e.,the gene of the invention) can be expressed by an arbitrary expressionpromoter and can be used for the gene therapy of cancers, for example.

1. Vectors

The transferable viral vectors may be prepared from DNA viruses or RNAviruses. They may be any viral vector of an MoMLV vector, a herpes virusvector, an Adenovirus vector, an AAV vector, a HIV vector, a Seidaivirus vector and the like. One or more proteins among the constituentprotein group of a viral vector are substituted by the constituentproteins of a different species of virus, or alternatively a part of thenucleic acid sequence constituting genetic information is substituted bythe nucleic acid sequence of a different species of virus to form aviral vector of the pseudo-type which can also be used in thisinvention. For example, there is mentioned a pseudo-type viral vectorwherein the Env protein (an envelop protein of HIV) is substituted bythe VSV-G protein (an envelop protein of vesicular stomatitis virus orVSV) (Naldini L., et al., Science 272, 263–267, 1996). Further, virueshaving a host spectrum other than human is usable as the viral vectorinsofar as they are efficacious. As for the vectors other than those ofviral origin, there may be used complexes of calcium phosphate andnucleic acid, ribosomes, cation-lipid complexes, Seidai virus liposomes,polymer carriers having polycation as the main chain and others. Inaddition, methods such as electroporation and gene guns may be used as agene transfer system.

2. Expression Promoters

As for the expression cassettes to be used for the therapeutic gene, anycassettes without any particular limitations may be used insofar as theycan cause genes to express in the target cells. One skilled in the artcan readily select such expression cassettes. Preferably, they areexpression cassettes capable of gene expression in the cells derivedfrom an animal, more preferably, expression cassettes capable of geneexpression in the cells derived from a mammal, and most preferablyexpression cassettes capable of gene expression in the cells derivedfrom a human. The gene promoters that can be used as expressioncassettes include: for example, virus-derived promoters from anAdenovirus, a cytomegalovirus, a human immunodeficiency virus, a simianvirus 40, a Rous sarcoma virus, a herpes simplex virus, a murineleukemia virus, a sinbis virus, a hepatitis type A virus, a hepatitistype B virus, a hepatitis type C virus, a papilloma virus, a human Tcell leukemia virus, an influenza virus, a Japanese encephalitis virus,a JC virus, parbovirus B19, a poliovirus, and the like; mammal-derivedpromoters such as albumin, SR α, a heat shock protein, and an elongationfactor; chimera type promoters such as a CAG promoter; and the promoterswhose expression can be induced by tetracyclines, steroids and the like.

The gene group discovered by this invention as being expressed in humanneuroblastomas with favorable prognosis will now be explained in greaterdetail by way of the examples; however, the technical scope of theinvention will not be restricted to those example.

EXAMPLES Production Example 1 Construction of cDNA Library from HumanNeuroblastoma

1. Obtaining Samples

Human neuroblastoma clinical tissue specimens were quasi-asepticallyfrozen immediately after surgical extraction and then preserved at −80°C.

2. Selecting Samples with Favorable Prognosis

Prognosis of the samples obtained in 1. above was carried out based onthe following criteria.

Favorable prognosis Unfavorable prognosis Stage 1 or 2 Stage 4 Age ofonset: <1 Age of onset: ≧1 Survival for ≧5 years Death within 3 yearsafter surgery without after surgery recurrence No amplification of N-mycAmplification of N-myc

Amplification of N-myc in the aforementioned two sample types wasconfirmed in the following manner.

The clinical tissue specimen obtained in 1. above was thinly sliced witha scalpel and then thoroughly homogenized after addition of 5 ml of TENbuffer (50 mM Tris-HCl (pH=8.0)/1 mM EDTA/100 mM NaCl). Upon adding 750μl of SDS (10%) and 125 μl of proteinase K (20 mg/ml) to the mixture, itwas gently stirred and allowed to stand at 50° C. for 8 hours. This wasfollowed by phenol/chloroform treatment and finally ethanolprecipitation to obtain purified genomic DNA. A 5 μg portion of theobtained genomic DNA was completely digested with the restrictionendonuclease EcoRI (NEB Inc.), and an N-myc probe was used to determineamplification of N-myc by Southern hybridization.

3. Preparation of mRNA from Clinical Tissue of Human Neuroblastoma withFavorable Prognosis

A 2–3 g portion of the clinical tissue samples of human neuroblastomajudged to have favorable prognosis in 2. above was treated using a TotalRNA Extraction Kit (QIAGEN Inc.) and the total RNA was extracted. Theextracted total RNA was purified using an oligo dT cellulose column(Collaborative Research, Inc.) to obtain a pool of mRNA with a polyAstructure.

4. Dephosphorylation of mRNA

A 100–200 μg portion of the mRNA pool prepared in 3. above was dissolvedin 67.3 μl of distilled sterile water containing 0.1% diethylpyrocarbonate (DEPC), and then 20 μl of 5×BAP buffer (Tris-HCl (500 mM,pH=7.0)/mercaptoethanol (50 mM)), 2.7 μl of RNasin (40 unit/μl: PromegaInc.) and 10 μl of BAP (0.25 unit/μl, bacteria-derived alkaliphosphatase: Takara Shuzo Co. Ltd.) were added. The mixture was reactedat 37° C. for 1 hour to effect dephosphorylation of the 5′ end of themRNA. This was followed by phenol/chloroform treatment two times, andfinally ethanol precipitation to obtain a purified dephosphorylated mRNApool.

5. Decapping of Dephosphorylated mRNA

The total amount of the dephosphorylated mRNA pool prepared in 4. abovewas dissolved in 75.3 μl of distilled sterile water containing 0.1%DEPC, and then 20 μl of 5×TAP buffer (sodium acetate (250 mM,pH=5.5)/mercaptoethanol (50 mM), EDTA (5 mM, pH=8.0)), 2.7 μl of RNasin(40 unit/μl) and 2 μl of TAP (tobacco acid pyrophosphatase:20 unit/μl)were added. The mixture was reacted at 37° C. for 1 hour to effectdecapping treatment of the 5′ end of the dephosphorylated mRNA. Thedephosphorylated mRNA of incomplete length with no capped structureremained without decapping, and with the 5′ end dephosphorylated. Thiswas followed by phenol/chloroform treatment and ethanol precipitation toobtain a purified decapped mRNA pool.

6. PreDaration of OliQo-Capped mRNA

The total amount of the decapped mRNA pool prepared in 5. above wasdissolved in 11 il of distilled sterile water containing 0.1% DEPC, andthen 4 il of 5′-oligo RNA (5′- AGCAUCGAGUCGGCCUUGGCCUACUGG-3′: 100ng/il) (SEQ ID NO: 105), 10 il of 10×ligation buffer (Tris-HCI (500 mM,pH=7.0)/mercaptoethanol (100 mM)), 10 il of magnesium chloride (50 mM),2.5 il of ATP (24 mM), 2.5il of RNasin (40 unit/il), 10 il of T4 RNAligase (25 unit/il: Takara Shuzo Co. Ltd.) and 50 il of polyethyleneglycol (50% w/v, PEG8000: Sigma Corporation) were added. The mixture wasreacted at 20° C. for 3 hours for ligation of the 5′-oligo RNA to the 5′end of the decapped mRNA. The dephosphorylated mRNA of incomplete lengthwith no capped structure resulted in no ligation to the 5′-oligo RNA.This was followed by phenol/chloroform treatment and ethanolprecipitation to obtain a purified oligo-capped mRNA pool.

7. Removal of DNA from Oligo-Capped mRNA

The oligo-capped mRNA pool prepared in 6. above was dissolved in 70.3 μlof distilled sterile water containing 0.1% DEPC, and then 4 μl ofTris-HCl (1 M, pH=7.0), 5.0 μl of DTT (0.1 M), 16 μl of magnesiumchloride (50 mM), 2.7 μl of RNasin (40 unit/μl) and 2 μl of DNaseI (5unit/μl: Takara Shuzo Co. Ltd.) were added. The mixture was reacted at37° C. for 10 minutes to dissolve the excess DNA. This was followed byphenol/chloroform treatment and ethanol precipitation and columnpurification (S-400HR: Pharmacia Biotech Inc.), to obtain a purifiedDNA(−) oligo-capped mRNA pool.

8. Preparation of 1st Strand cDNA

The DNA(−) oligo-capped mRNA pool prepared in 7. above was reversetranscribed using SuperScript II (kit by Life Tech Oriental, Inc.) toobtain a pool of 1st strand cDNA. The pool of DNA(−) oligo-capped mRNAwas dissolved in 21 il of sterile distilled water, and then 10 il of 10×First Strand buffer (kit accessory), 8 il of dNTP mix (5 mM, kitaccessory), 6 il of DTT (0.1 M, kit accessory), 2.5 il of oligo-dTadapter primer (5 pmol/il, 5′-GCGGCTGAAGACGGCCTATGTGGCCTTTTTTTTTTTTTTTTT-3′), (SEQ ID NO: 106) 2.0 il of RNasin (40 unit/il)and 2 il of SuperScript II RTase (kit accessory) were added. The mixturewas reacted at 42° C. for 3 hours to effect reverse transcription. Thiswas followed by phenol/chloroform treatment, alkali treatment andneutralization treatment to dissolve all the RNA and purification wascarried out by ethanol precipitation.

9. Preparation of 2nd Strand cDNA

The 1st strand cDNA pool prepared in 8. above was subjected to PCRamplification using Gene Amp (kit by Perkin Elmer Inc.). The pool of 1ststrand cDNA was dissolved in 52.4 il of sterile distilled water, andthen 30 il of 3.3× Reaction buffer (kit accessory), 8 il of dNTP mix(2.5 mM, kit accessory), 4.4 il of magnesium acetate (25 mM, kitaccessory), 1.6 il of Primer F (10 pmol/il,5′-AGCATCGAGTCGGCCTTGTTG-3′), (SEQ ID NO: 107), 1.6 il of Primer R (10pmol/il, 5′-GCGCTGAAGACGGCCTATGT-3′) (SEQ ID NO: 108) and 2 il of rTth(kit accessory) were added. A 100 il portion of mineral oil was gentlyadded to the mixture and overlayed thereon. After denaturing thereaction solution at 94° C. for 5 minutes, a cycle of 94° C. for 1minute, 52° C. for 1 minute and 72° C. for 10 minutes was repeated 12times, and then the solution was allowed to stand at 72° C. for 10minutes to complete the PCR reaction. This was followed byphenol/chloroform treatment and ethanol precipitation to obtain a 2ndstrand cDNA pool.

10. SfiI Treatment of 2nd Strand cDNA

The 2nd strand cDNA pool prepared in 9. above was dissolved in 87 μl ofsterile distilled water, and then 10×NEB buffer (NEB Inc.), 100×BSA(bovine serum albumin available from NEB Inc.) and 2 μl of SfiI(restriction endonuclease, 20 unit/μl, NEB Inc.) were added. The mixturewas reacted overnight at 50° C. to effect SfiI restriction endonucleasetreatment. This was followed by phenol/chloroform treatment and ethanolprecipitation to obtain a pool of cDNA which had been SfiI-treated atboth ends.

11. Size Fractionation of SfiI-Treated cDNA

The SfiI-treated cDNA pool prepared in 10. above was electrophoresed on1% agarose gel and a fraction with >2 kb was purified using Geneclean II(Bio101 Inc.). The purified cDNA pool was dissolved in 100 μl of steriledistilled water and allowed to stand at 37° C. for 6 hours. This wasfollowed by phenol/chloroform treatment and ethanol precipitation toobtain a long-chain cDNA pool.

12. cDNA Library

The long-chain cDNA pool prepared in 11. above was ligated into thecloning vector pME18S-FL3 (provided by Prof. Sumio Kanno of theInstitute of Medical Science, Tokyo University) using a DNA Ligation Kitver. 1 (kit by Takara Shuzo Co. Ltd.). The long-chain cDNA pool wasdissolved in 8 μl of sterile distilled water, and then 1 μl ofpME18S-FL3 pretreated with restriction endonuclease DraIII, 80 μl ofSolution A (kit accessory) and 10 μl of Solution B (kit accessory) wereadded and reaction was conducted at 16° C. for 3 hours. This wasfollowed by phenol/chloroform treatment and ethanol precipitation forpurification to obtain a cDNA library.

Example 2 Transformation into E. coli

1. Cloning

The cDNA library prepared in Example 1, 12. above was used fortransformation into E. coli (TOP-10: Invitrogen Corporation). The cDNAlibrary was dissolved in 10 μl of sterile distilled water and mixed withTOP-10. The mixture was then incubated on ice for 30 minutes, at 40° C.for 1 minute and on ice for 5 minutes. After adding 500 μl of SOBmedium, shake culturing was performed at 37° C. for 60 minutes.Appropriate amounts thereof were seeded onto ampicillin-containing agarmedia and culturing was continued at 37° C. for a day and a night toobtain E. coli clones.

2. Preservation of E. coli Clones (Preparation of Glycerol Stock)

The E. coli clones on agar media obtained in 1. above were collectedwith toothpick and suspended in 120 μl of LB medium prepared in a96-well plate. The 96-well plate was then allowed to stand overnight at37° C. for culturing of the E. coli. A 72 μl portion of 60% glycerolsolution was then added and preserved at −20° C. (glycerol stock)

Example 3 Nucleic Acid Sequence Determination

1. Preparation of Plasmid

The 10 μl of glycerol stock prepared in Example 2, 2. above wastransferred to a 15 ml centrifugation tube, and then 3 ml of LB mediumand 50 g g/ml of ampicillin were added and shaking was carried outovernight at 37° C. for culturing of the E. coli. A QIAprep SpinMiniprep Kit (QIAGEN Inc.) was then used to extract and purify a plasmidDNA from the E. coli.

2. Analysis of Both End Sequences

Both end sequences of the plasmid DNA prepared in 1. above weredetermined using a DNA Sequencing Kit (kit by ABI). There were combined600 ng of plasmid DNA, 8 μl of premix (kit accessory) and 3.2 pmol ofprimers, and sterile distilled water was added to a total of 20 μl Afterdenaturing the mixture at 96° C. for 2 minutes, a cycle of 96° C. for 10seconds, 50° C. for 5 seconds and 60° C. for 4 minutes was repeated 25times for reaction. The product was then purified by ethanolprecipitation. Sequence determination was carried out by polyacrylamidegel electrophoresis under denaturing conditions, using ABI377 (ABI).

Example 4

Homology Search of Database

An internet nucleic acid sequence homology search was conducted for thenucleic acid sequence data obtained from the both end-sequence analysisin Example 3. The search was conducted using the BLAST database of theNCBI (National Center of Biotechnology Information,http://www.ncbi.nblm.nih.gov/BLAST).

Example 5

Comparison of Gene Expression Levels in Human Neuroblastomas withFavorable Prognosis and Unfavorable Prognosis by Semi-Quantitative PCR

PCR primers were synthesized from the nucleic acid sequences of portionsof the gene group obtained in Example 4, and the expression levels inthe clinical tissues of human neuroblastomas with favorable prognosisand unfavorable prognosis were comparatively quantitated. mRNA wasextracted from the human neuroblastoma clinical tissues by the methoddescribed in Examples 1–3, and rTaq (Takara Shuzo Co. Ltd.) was used forPCR reaction. Specifically, 5 μl of sterile distilled water, 2 μl ofmRNA, 1 μl of 10×rTaq buffer, 1 μl of 2 mM dNTPs, 0.5 μl each of thesynthesized primer set and 0.5 μl of rTaq were combined. Afterdenaturing the mixture at 95° C. for 2 minutes, a cycle of 95° C. for 15seconds, 55° C. for 15 seconds and 72° C. for 20 seconds was repeated 35times, and then the mixture was allowed to stand at 72° C. for 6 minutesfor PCR reaction. The reaction solution was subjected to 1% agarose gelelectrophoresis. Consequently, when the PCR primers based on the nucleicacid sequences set forth in SEQ ID NO:1 to NO:104 in the SequenceListing were used in amplification, the genes whose expression wasenhanced only in neuroblastomas with favorable prognosis wereidentified. Tables 1 and 2 show the information on the nucleic acidsequences set forth in SEQ ID NO:1 to NO:104, including the results ofthe homology search shown in Example 4 (73 nucleic acid sequences among104 nucleic acid sequences had no homology).

Examples of the measurements of gene expression levels in humanneuroblastomas with favorable prognosis and unfavorable prognosis bysemi-quantitative PCR (Nucleic acid sequences nbla-00106, nbla-00219 andnbla-03145) are shown in FIGS. 1–3.

TABLE 1 Nucleic acid sequences whose expression is enhanced inneuroblastomas with favorable prognosis CELL CLONE CYCLE PHASE HOMOLOGYSEQ ID NAME SPECIFICITY (ACCESSION No.) 1 nbla-00002 KIAA0327(AB002325)2 nbla-00012 S PHASE — 3 nbla-00052 — 4 nbla-00067 — 5 nbla-00078 SPHASE KIAA0322(AB002320) 6 nbla-00086-f GTPaseRAB6B(AF166492) 7nbla-00086-r — 8 nbla-00100 G2/M PHASE KIAA0632(AB014532) 9 nbla-00106 —10 nbla-00113 KIAA0874(AB020681) 11 nbla-00118 — 12 nbla-00126MAB21L1(NM_005584) 13 nbla-00137 — 14 nbla-00150 G2/M PHASESART-3(AB020880) 15 nbla-00158 — 16 nbla-00172 G2/M PHASE — 17nbla-00177 S PHASE — 18 nbla-00204 — 19 nbla-00219 KIAA0367(AB002365) 20nbla-00235 G2/M PHASE — 21 nbla-00237 — 22 nbla-00271 KIAA0886(AB020693)23 nbla-00343 KIAA1145(AB032971) 24 nbla-00371 S PHASE — 25 nbla-00375 —26 nbla-00418 — 27 nbla-00433 — 28 nbla-00437 S PHASE AND — G2/M PHASE29 nbla-00490 G2/M PHASE T1-227H(D50525) 30 nbla-00538-fDKFZp586D1146(AL080222) 31 nbla-00538-r DKFZp566D1146(AL080222) 32nbla-00613 — 33 nbla-00650 — 34 nbla-00652 S PHASE AND FLJ10739fis(AK001601) G2/M PHASE 35 nbla-00660 G2/M PHASE — 36 nbla-00693DKFZp434G0827(AL122107) 37 nbla-00697 G1 PHASE AND — S PHASE 38nbla-00715 — 39 nbla-00744 — 40 nbla-00761 S PHASE KIAA0751(AB018294) 41nbla-00830-f — 42 nbla-00830-r — 43 nbla-00831-f KIAA0868(AB020675) 44nbla-00831-r KIAA0868(AB020675) 45 nbla-00832-f — 46 nbla-00832-r(AF140710) 47 nbla-02942 (NM_001788) 48 nbla-02975 G1 PHASE FLJ10103fis(AK000965) 49 nbla-02981 — 50 nbla-02999 G2/M PHASE (AF182814) 51nbla-03010 G1 PHASE — 52 nbla-03103 G1 PHASE — 53 nbla-03107-fKIAA1309(AB037730) 54 nbla-03107-r KIAA1309(AB037730)

TABLE 2 Nucleic acid sequences whose expression is enhanced inneuroblastomas with favorable prognosis 55 nbla-03139 S PHASE ANDFOG2(NM_012082) M PHASE 56 nbla-03145 G1 PHASE XCE(Y16187) 57nbla-03199-f S PHASE — 58 nbla-03199-r S PHASE — 59 nbla-03212-f S PHASE— 60 nbla-03212-r S PHASE — 61 nbla-03219-f — 62 nbla-03219-r — 63nbla-03301-f S PHASE NF-L(X05608) 64 nbla-03301-r S PHASE — 65nbla-03461-f — 66 nbla-03461-r — 67 nbla-03539-f S PHASE — 68nbla-03539-r S PHASE — 69 nbla-03575-f S PHASE AND KIAA0517(AB011089)G2/M PHASE 70 nbla-03575-r S PHASE AND — G2/M PHASE 71 nbla-03646-fKIAA0018(D13643) 72 nbla-03646-r KIAA0018(D13643) 73 nbla-03684-f — 74nbla-03755-r S PHASE — 75 nbla-03759-f — 76 nbla-03759-r — 77nbla-03761-f — 78 nbla-03761-r — 79 nbla-03771-f — 80 nbla-03771-r — 81nbla-03777-f — 82 nbla-03777-r — 83 nbla-03779-f — 84 nbla-03779-r — 85nbla-03781-f — 86 nbla-03781-r DKFZp434C035(AL137633) 87 nbla-03831-f —88 nbla-03831-r — 89 nbla-03851-f — 90 nbla-03851-r — 91 nbla-03862-f —92 nbla-03862-r — 93 nbla-03898-f — 94 nbla-03898-r — 95 nbla-03911-f —96 nbla-03911-r — 97 nbla-03914-f — 98 nbla-03914-r — 99 nbla-04021-f —100 nbla-04021-r — 101 nbla-04055-f — 102 nbla-04055-r — 103nbla-04061-f — 104 nbla-04061-r —

Example 6

Measurement of Cell Cycle Phase-Dependent Gene Expression Levels bySemi-Quantitative PCR

PCR primers were synthesized from the nucleic acid sequences of portionsof the gene group obtained in Example 4, and HeLa cells were used forcomparative quantitation of cell cycle phase-dependent gene expressionlevels. The HeLa cells used were treated in each of the followingmanners.

-   (1) Untreated-   (2) Treated with 400 μM of mimosine for 18 hours, with 65% of the    cells arrested in the Gl phase.-   (3) Treated with 2 mM thymidine for 20 hours, with 100% of the cells    arrested in the S phase.-   (4) Treated with 0.6 μg/ml of nocodazole, with 85% of the cells    arrested in the G2/M phase.    mRNA was extracted from the aforementioned 4 types of HeLa cells by    the method described in Examples 1–3, and rTaq (Takara Shuzo Co.    Ltd.) was used for PCR reaction. Specifically, 5 μl of sterile    distilled water, 2 μl of mRNA, 1 μl of 10×rTaq buffer, 1 μl of 2 mM    dNTPs, 0.5 μl each of the synthesized primer set and 0.5 μl of rTaq    were combined. After denaturing the mixture at 95° C. for 2 minutes,    a cycle of 95° C. for 15 seconds, 55° C. for 15 seconds and 72° C.    for 20 seconds was repeated 35 times, and then the mixture was    allowed to stand at 72° C. for 6 minutes for PCR reaction. The    reaction solution was subjected to 1% agarose gel electrophoresis.    Consequently, when the PCR primers based on the nucleic acid    sequences set forth in SEQ ID NO:1 to NO:104 in the Sequence Listing    were used in amplification, it was found that the gene expression    was specific for cell cycle phase in 31 nucleic acid sequences. An    example of the electrophoresis results (Nucleic acid sequence    nbla-00100) is shown in FIG. 4. Also, Tables 1 and 2 have displayed    a tabulation of the cell cycle phase specificities and individual    nucleic acid sequences that were discovered in the manner presented    herein.

INDUSTRIAL APPLICABILITY

The nucleic acids of this invention provide information relating to thegenes expressed in neuroblastoma.

The nucleic acids of the invention or their fragments may be used asprobes or primers for various types of hybridization or PCR, and permitdetection of the expression of the aforementioned genes in other tissuesand cells, as well as analysis of their structure and functions.Production of the human proteins encoded by the genes through geneticengineering is also possible.

The nucleic acids of the invention are those derived from a gene whoseexpression is enhanced in human neuroblastoma with favorable prognosisbased on comparison between human neuroblastoma with favorable prognosisand human neuroblastoma with unfavorable prognosis, and therefore allowthe diagnosis for prognosis of neuroblastoma based on this geneticinformation from these nucleic acids. Unlike the N-myc gene which is afactor for unfavorable prognosis, these genes are considered factors forfavorable prognosis, similar to the TrkA gene, and therefore can serveas markers (tumor markers) for neuroblastoma malignancy and sensitivityto anti-cancer agents.

1. An isolated nucleic acid comprising a sequence selected from thegroup consisting of nucleic acid sequences set forth in SEQ ID NO:3, SEQID NO:5, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:32, SEQ IDNO:40, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:74, in the SequenceListing or a complementary nucleic acid thereof.
 2. The nucleic acidaccording to claim 1, wherein the nucleic acid is DNA.
 3. The nucleicacid according to claim 1, wherein the nucleic acid is derived from agene expressed in human neuroblastoma.
 4. A method of diagnosising theprognosis of human neuroblastoma, said method comprising extracting aneuroblastoma specimen from a subject; detecting at least one nucleicacid in the specimen, the nucleic acid comprising a sequence selectedfrom the group consisting of nucleic acid sequences set forth in SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:22, SEQ IDNO:32, SEQ ID NO:40, SEQ ID NO:55, SEQ ID NO:56 and SEQ ID NO:74, in theSequence Listing or a complementary nucleic acid thereof; and diagnosingthe prognosis of the human neuroblastoma as favorable if said nucleicacid is detected.
 5. The isolated nucleic acid according to claim 1,wherein the nucleic acid sequence is SEQ ID NO:3 in the SequenceListing.
 6. The isolated nucleic acid according to claim 1, wherein thenucleic acid sequence is SEQ ID NO:5 in the Sequence Listing.
 7. Theisolated nucleic acid according to claim 1, wherein the nucleic acidsequence is SEQ ID NO:9 in the Sequence Listing.
 8. The isolated nucleicacid according to claim 1, wherein the nucleic acid sequence is SEQ IDNO:19 in the Sequence Listing.
 9. The isolated nucleic acid according toclaim 1, wherein the nucleic acid sequence is SEQ ID NO:22 in theSequence Listing.
 10. The isolated nucleic acid according to claim 1,wherein the nucleic acid sequence is SEQ ID NO:32 in the SequenceListing.
 11. The isolated nucleic acid according to claim 1, wherein thenucleic acid sequence is SEQ ID NO:40 in the Sequence Listing.
 12. Theisolated nucleic acid according to claim 1, wherein the nucleic acidsequence is SEQ ID NO:55 in the Sequence Listing.
 13. The nucleic acidaccording to claim 1, wherein the nucleic acid sequence is SEQ ID NO:56in the Sequence Listing.
 14. The isolated nucleic acid according toclaim 1, wherein the nucleic acid sequence is SEQ ID NO:74 in theSequence Listing.